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Abstract：
OBJECTIVE: To study the molecular pathogenesis of PAPA (pyogenic arthritis, pyoderma gangrenosum and acne) syndrome, a debilitating hereditary autoinflammatory disease caused by dominant mutation in PSTPIP1.
METHODS: Gene knock-out and knock-in mice were generated to develop an animal model. THP1 and retrovirally transduced U937 human myeloid leukaemia cell lines, peripheral blood mononuclear cells, small interfering RNA (siRNA) knock-down, site-directed mutagenesis, cytokine immunoassays, coimmunoprecipitation and immunoblotting were used to study inflammasome activation. Cytokine levels in the skin were evaluated by immunohistochemistry. Responsiveness to Janus kinase (JAK) inhibitors was evaluated ex vivo with peripheral blood mononuclear cells and in vivo in five treatment-refractory PAPA patients.
RESULTS: The knock-in mouse model of PAPA did not recapitulate the human disease. In a human myeloid cell line model, PAPA-associated PSTPIP1 mutations activated the pyrin inflammasome, but not the NLRP3, NLRC4 or AIM2 inflammasomes. Pyrin inflammasome activation was independent of the canonical pathway of pyrin serine dephosphorylation and was blocked by the p.W232A PSTPIP1 mutation, which disrupts pyrin-PSTPIP1 interaction. IFN-γ priming of monocytes from PAPA patients led to IL-18 release in a pyrin-dependent manner. IFN-γ was abundant in the inflamed dermis of PAPA patients, but not patients with idiopathic pyoderma gangrenosum. Ex vivo JAK inhibitor treatment attenuated IFN-γ-mediated pyrin induction and IL-18 release. In 5/5 PAPA patients, the addition of JAK inhibitor therapy to IL-1 inhibition was associated with clinical improvement.
CONCLUSIONS: PAPA-associated PSTPIP1 mutations trigger a pyrin-IL-18-IFN-γ positive feedback loop that drives PAPA disease activity and is a target for JAK inhibition.
METHODS: Gene knock-out and knock-in mice were generated to develop an animal model. THP1 and retrovirally transduced U937 human myeloid leukaemia cell lines, peripheral blood mononuclear cells, small interfering RNA (siRNA) knock-down, site-directed mutagenesis, cytokine immunoassays, coimmunoprecipitation and immunoblotting were used to study inflammasome activation. Cytokine levels in the skin were evaluated by immunohistochemistry. Responsiveness to Janus kinase (JAK) inhibitors was evaluated ex vivo with peripheral blood mononuclear cells and in vivo in five treatment-refractory PAPA patients.
RESULTS: The knock-in mouse model of PAPA did not recapitulate the human disease. In a human myeloid cell line model, PAPA-associated PSTPIP1 mutations activated the pyrin inflammasome, but not the NLRP3, NLRC4 or AIM2 inflammasomes. Pyrin inflammasome activation was independent of the canonical pathway of pyrin serine dephosphorylation and was blocked by the p.W232A PSTPIP1 mutation, which disrupts pyrin-PSTPIP1 interaction. IFN-γ priming of monocytes from PAPA patients led to IL-18 release in a pyrin-dependent manner. IFN-γ was abundant in the inflamed dermis of PAPA patients, but not patients with idiopathic pyoderma gangrenosum. Ex vivo JAK inhibitor treatment attenuated IFN-γ-mediated pyrin induction and IL-18 release. In 5/5 PAPA patients, the addition of JAK inhibitor therapy to IL-1 inhibition was associated with clinical improvement.
CONCLUSIONS: PAPA-associated PSTPIP1 mutations trigger a pyrin-IL-18-IFN-γ positive feedback loop that drives PAPA disease activity and is a target for JAK inhibition.
摘要：
目的：研究PAPA（化脓性关节炎，坏疽性脓皮病和痤疮）综合征，由PSTPIP1显性突变引起的一种使人衰弱的遗传性自身炎症性疾病。
方法:产生基因敲除和敲入小鼠以建立动物模型。THP1和逆转录病毒转导的U937人髓系白血病细胞系，外周血单核细胞，小干扰RNA（siRNA）敲低，定点诱变，细胞因子免疫测定，共免疫沉淀和免疫印迹用于研究炎性体激活。通过免疫组织化学评估皮肤中的细胞因子水平。对Janus激酶（JAK）抑制剂的反应进行了外周血单核细胞的离体评估，并在五名治疗难治性PAPA患者中进行了体内评估。
结果:PAPA的敲入小鼠模型没有概括人类疾病。在人类骨髓细胞系模型中,PAPA相关的PSTPIP1突变激活了pyrin炎性体，但不是NLRP3，NLRC4或AIM2炎性体。Pyrin炎性体激活独立于pyrin丝氨酸去磷酸化的经典途径，并被p.W232APSTPIP1突变阻断，这破坏了pyrin-PSTPIP1的相互作用。来自PAPA患者的单核细胞的IFN-γ引发导致IL-18以pyrin依赖性方式释放。IFN-γ在PAPA患者发炎的真皮中含量丰富，但不是特发性坏疽性脓皮病患者。离体JAK抑制剂治疗减弱了IFN-γ介导的吡喃蛋白诱导和IL-18释放。在5/5PAPA患者中，在IL-1抑制的基础上增加JAK抑制剂治疗与临床改善相关.
结论：PAPA相关的PSTPIP1突变触发pyrin-IL-18-IFN-γ正反馈回路，驱动PAPA疾病活动，是JAK抑制的靶标。
方法:产生基因敲除和敲入小鼠以建立动物模型。THP1和逆转录病毒转导的U937人髓系白血病细胞系，外周血单核细胞，小干扰RNA（siRNA）敲低，定点诱变，细胞因子免疫测定，共免疫沉淀和免疫印迹用于研究炎性体激活。通过免疫组织化学评估皮肤中的细胞因子水平。对Janus激酶（JAK）抑制剂的反应进行了外周血单核细胞的离体评估，并在五名治疗难治性PAPA患者中进行了体内评估。
结果:PAPA的敲入小鼠模型没有概括人类疾病。在人类骨髓细胞系模型中,PAPA相关的PSTPIP1突变激活了pyrin炎性体，但不是NLRP3，NLRC4或AIM2炎性体。Pyrin炎性体激活独立于pyrin丝氨酸去磷酸化的经典途径，并被p.W232APSTPIP1突变阻断，这破坏了pyrin-PSTPIP1的相互作用。来自PAPA患者的单核细胞的IFN-γ引发导致IL-18以pyrin依赖性方式释放。IFN-γ在PAPA患者发炎的真皮中含量丰富，但不是特发性坏疽性脓皮病患者。离体JAK抑制剂治疗减弱了IFN-γ介导的吡喃蛋白诱导和IL-18释放。在5/5PAPA患者中，在IL-1抑制的基础上增加JAK抑制剂治疗与临床改善相关.
结论：PAPA相关的PSTPIP1突变触发pyrin-IL-18-IFN-γ正反馈回路，驱动PAPA疾病活动，是JAK抑制的靶标。
